To standardize a procedure for the enzymatic assay of Deoxyribonuclease I.
This procedure applies to all products that have a specification for Deoxyribonuclease I activity.
3.1. Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC
3.2. Unit Definition - One Kunitz unit will produce a ΔA260 of 0.001 per minute per mL at pH 5.0 at 25 ºC, using DNA Types I or III as substrate.
3.3. DNA - Deoxyribonucleic Acid
DNA + H2O Deoxyribonuclease I > 5- Oligodeoxy ribonucleotides
There is no absolute standard for the assay of Deoxyribonuclease. When the procedure of Kunitz is used the result is affected by the particular lot of substrate that is used. For DNase studies, Sigma-Aldrich offers a standardized vial, (D4263), which has been standardized to contain 2,000 Kunitz units using our DNA (D3664) as a substrate.
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions. Deoxyribonuclease I is a sensitizer, appropriate care should be taken to minimize exposure.
7.1. CONDITIONS: T = 25 ºC, pH = 5.0, A260nm, Light Path = 1cm
7.2. METHOD: Continuous Spectrophotometric Rate Determination
7.3.1. 1.0 M Sodium Acetate Buffer, pH 5.0 at 25 ºC (Buffer) Prepare a 136 mg/mL solution in purified water using Sodium Acetate, Trihydrate (S8625). Adjust to pH 5.0 at 25 ºC with 5 M HCl.
7.3.2. 100mM Magnesium Sulfate Solution (MgSO4) Prepare a 12 mg/mL solution in purified water using Magnesium Sulfate, Heptahydrate (M1880).
7.3.3. 0.033% (w/v) Deoxyribonucleic Acid Solution (DNA)
18.104.22.168. Prepare in purified water using Deoxyribonucleic Acid (D3664).
22.214.171.124. Dissolve at a concentration of 0.33 mg DNA/mL. Let the solution stand on ice for a minimum of 30 minutes then mix by slow inversion until completely dissolved.
126.96.36.199. If multiple vials of D3664 are used, prepare each separately and then combine the contents before continuing with Assay Procedure step 7.4.1.
7.3.4. 0.85% Sodium Chloride (NaCl)
Use Sodium Chloride Solution, 0.85%, (S0817).
7.3.5. Standard DNase Solution (Std Soln)
188.8.131.52. Reconstitute a vial of Deoxyribonuclease I, Standardized vial, containing 2000 Kunitz units, (D4263), with 1 mL of cold Reagent 7.3.4 (NaCl).
184.108.40.206. Immediately before use, dilute to 400-500 uncorrected Kunitz units/mL with cold Reagent 7.3.4 (NaCl). The uncorrected units/mL corresponds to ΔA260 nm/min of 0.067 – 0.083.
7.3.6. Deoxyribonuclease I Enzyme Solution (Test)
220.127.116.11. Immediately before use, prepare a solution containing 400 - 500 Kunitz units/mL in cold Reagent 7.3.4 (NaCl).
18.104.22.168. Samples for impurity testing should be run at 10 mg/mL unless otherwise specified.
7.4. ASSAY PROCEDURE
7.4.1. Determine the concentration of DNA in Reagent 7.3.3 by the following:
22.214.171.124. Pipette the following (in milliliters) into suitable quartz cuvettes (perform in duplicate):
126.96.36.199. Record the absorbance of each Test at 260nm. This is the Blank. Then add the following to each cuvette:
188.8.131.52. Record the absorbance of each Test at 260nm. This is the Test.
184.108.40.206. Calculate the DNA concentration by the following:
|mg DNA/mL =||(A260nm Test - A260nm Blank) X 3.0
|20 X 0.1|
20 = E0.1% of DNA. One A260nm unit is equivalent to 50 micrograms of DNA. 3.0 = final volume of test solution
7.4.2. Prepare a reaction cocktail by pipetting the following (in milliliters) into a suitable container:
*Note: The amounts of DNA and water in the reaction cocktail may be adjusted as necessary due to the concentration of Reagent 7.3.3. The final concentration of DNA in the reaction cocktail should be 0.004%(w/v) or 0.04mg/mL.
7.4.3. Gently mix by swirling and then adjust the pH of this solution to 5.0 at 25 ºC with 0.1N HCl or NaOH, as necessary.
7.4.4. Pipette the following (in milliliters) into suitable quartz cuvettes:
7.4.5. Equilibrate at 25 ºC in a suitably thermostatted spectrophotometer for approximately 5 minutes, then add:
220.127.116.11 If varying levels of enzyme are being run, the total reaction volume must be adjusted to 3.00 mL using Reagent 7.3.4. (NaCl).
18.104.22.168 The fastest rate is usually in the first 1-2 minutes of the reaction. It is, therefore, advisable to run only one or two reactions at a time.
7.4.6. Immediately mix by inversion and record the increase in A260nm for approximately 3-5 minutes. Obtain the ΔA260nm/minute using the maximum linear rate for the Blank, Standards and Tests.
|Units/ Std Vial =||(ΔA260nm/min Std - ΔA260nm/min Blank) X (3) X (df)
|(0.001) X (0.5)|
|Correction Factor =||Release value of Std vial(Units/vial)
|Experimental values/Std vial|
|Units/ mg solid =||(ΔA260nm/min Test- ΔA260nm/min Blank) X (3) X (df) X (CF)
|(0.001) X (0.5)|
3 = Volume (milliliters) of assay
0.5 = volume of enzyme used in each test
df = dilution factor
0.001 = ΔA260nm per the unit definition
CF = Correction Factor
7.6. FINAL ASSAY CONCENTRATION
In a 3.00 mL reaction mix, the final concentrations are 83mM Sodium Acetate, 4.2mM Magnesium Sulfate, 0.14% Sodium Chloride, 0.003% (w/v) Deoxyribonucleic Acid and 200-250 units of Deoxyribonuclease I.
8.1 Kunitz, M. (1950) Journal of General Physiology 33, 349-362
8.2 Kunitz, M. (1950) Journal of General Physiology 33, 363-377
8.3 Lindberg, U. (1964) Biochimica et Biophysica Acta 82, 237-248
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